1. Field of the Invention
The present invention relates to an apparatus and a method for reading a gel electrophoresis pattern and, more particularly, to a gel electrophoresis pattern reading apparatus and a gel electrophoresis pattern reading method so adapted as to read a gel electrophoresis pattern with high sensitivity to detection of the electrophoresis pattern without requiring any expensive device configuration such as a special laser light source, the gel electrophoresis pattern being obtained by subjecting a sample containing nucleic acids or protein acids to electrophoresis in gel and separating the sample from the gel material.
2. Description of the Related Art
Heretofore, techniques for analysis by means of gel electrophoresis methods have been extensively utilized for the fragmentation and for the analysis of structures of protein acids and nucleic acids present as a polymer in the body of an animal or plant. The gel electrophoresis method has the advantage that a minute amount of a sample can be analyzed in an appropriate manner so that in many cases the gel electrophoresis method is utilized for experiments in which a sample can be procured in a very limited amount. Therefore, the analysis using the gel electrophoresis method requires a very high sensitivity to detection of the sample.
In conventional techniques, a sample to be analyzed is first labelled with a radioactive isotope, the labelled sample is injected into a gel, and the resulting gel material is subjected to electrophoresis. After the gel electrophoresis, the gel is attached to an X-ray film or the like for exposure to X rays to be emitted from the radioactive isotope labelled in the sample. After exposure of the gel material containing the labelled sample to the X-ray film or the like for a given period of time, the X-ray film is then developed, and the exposed pattern resulting from the radioactive isotope transcribed onto the X-ray film is read as a gel electrophoresis pattern in order to analyze the structures of the proteins or nucleic acids of the sample.
The radioactive isotopes, however, are so hazardous that they should be handled and managed with extreme care and with high security. Recently, techniques for handling laser light and technologies of laser light sources, optical sensors and signal processing have greatly developed leading to the development of fluorescence detecting methods for detecting an electrophoresis pattern without requiring the use of such hazardous radioactive isotopes. The fluorescence detecting methods comprise labelling a sample with a fluorescent substance, subjecting the sample to electrophoresis, exciting the labelled fluorescent substance directly with a laser light source, and detecting the resulting fluorescent pattern, thereby resulting in the detection of the electrophoresis pattern.
As an example of reading an electrophoresis pattern by the fluorescence detecting method, which has initially been developed, there is known a method for determining the sequence of a DNA as disclosed in Japanese Patent Unexamined Publication Kokai No. 61-173,158. An outline of a fluorescence detecting method utilized for the DNA sequence determining method will be described with reference to FIGS. 4 and 5, showing a schematic representation of the method for the detection of a gel electrophoresis pattern by the fluorescence detecting method, in which FIG. 4 is a block diagram showing an outline of a gel electrophoresis apparatus and FIG. 5 shows the details of a portion of a fluorescence detecting section of the gel electrophoresis apparatus of FIG. 4.
First, a description will be made of the device structure of the gel electrophoresis apparatus with reference to FIG. 4. The gel electrophoresis apparatus comprises a gel material 31 in which electrophoresis is to be conducted, a fine tube 32 for retaining the gel material 31, an upper buffer solution container or tank 33 and a lower buffer solution container or tank 34, between which an electrical field is applied to the gel material 31 held in the fine tube 32, a first electrode 35a, a second electrode 35b, a light source 36 for exciting a fluorescent substance labelled in the electrophoresed sample, a detector 37 for detecting the fluorescence emitted from the sample, a data processing section 38 for processing signals transmitted from the detector 37 and converting the fluorescent signals into electrical signals, and an electric power source 39 for applying the electrophoresis electrical field between the first electrode 35a and the second electrode 35b. 
Next, an operation of the gel electrophoresis apparatus will be described by example where a DNA sample is electrophoresed as an object of electrophoresis and an electrophoresis pattern of the DNA sample is read with the gel electrophoresis apparatus. The DNA sample is first labelled with a fluorescent substance and the labelled sample is poured into the upper buffer solution container 33 from which the sample in turn is introduced into the fine tube 32, and an electrophoresis voltage of from several kV to approximately 10 kV is applied from the electric power source 39 between the first and second electrodes 35a and 35b. As the DNA has negative charges, they migrate toward the positive electrode of the second electrode 35b upon application of such electrophoresis voltage and they eventually reach the position of the light source 36. Thereafter, the fluorescent substance labelled in the DNA sample is excited in this position upon exposure to laser beams emitted from the light source 36, thereby emitting fluorescence that in turn is detected and received by the detector 37. The fluorescence received by the detector 37 is then converted into electrical signals and the detector 37 transmits the electrical signals to the data processing section 38 which in turn processes the electrical signals and determines the sequences of the DNA fragments separated by their molecular weights, thereby yielding an electrophoresis pattern.
The detector 37 is arranged such that the fluorescence emitted from the sample in the gel material 31 migrating within the fine tube 32 can be received in a manner as shown in FIG. 5, which is a partially transverse sectional view (looking down). As shown in FIG. 5, when the sample migrates downward through the gel material 31 and the sample reaches the position of the fine tube 32 which is irradiated with laser beams 40 emitted from the light source 36, the fluorescent substance labelled in the DNAs of the sample is excited with the laser beams 40, thereby resulting in the emission of fluorescence 41 that in turn is received by the detector 37. The received fluorescence 41 is then transmitted to a photomultiplier of the detector 37 and the photomultiplier converts the fluorescence 41 into electrical signals and transmits the electrical signals to the data processing unit 38. The data processing unit 38 is arranged such that the sequences of the DNA fragments in the sample are determined by the molecular weights on the basis of the peak positions of the intensity of the fluorescence 41 emitted from the DNA sample and received by the detector 37.
When they are employed as a sample, DNA fragments are labelled with the fluorescent substance so as to have different fluorescent wavelengths corresponding to their ingredients, i.e. four bases comprising adenine, cytosine, guanine and thymine, and to determine the DNA sequences of the four bases simultaneously by causing the DNA sample to migrate down through only one fine tube. The fluorescent substance with which to label the DNA fragments, which can emit four different fluorescent wavelengths, may include, for example, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (EITC), tetramethylrhodamine isothiocyanate (TMRITC), and substituted rhodamine isothiocyanate (XRITC), respectively. Further, the gel electrophoresis apparatus of this type has a sensitivity to detection of DNAs in the order of 1xc3x9710xe2x88x9216 mole as high as the method using radioactive isotopes, when argon ion laser having a wavelength of 488 nm or 514 nm is employed.
In addition, Japanese Patent Unexamined Publication Kokai No. 61-173,158 briefly alludes to an example in which luminescence can be caused by taking advantage of chemical reaction energy. The example does not specifically disclose any procedures of the elution from a gel material, a reaction with a luminous substance, the removal of an unnecessary labelling substance, and the like. Actually, many specific problems should be solved in embodying techniques utilizing luminescence based on the chemical reaction energy in a device structure. Such problems may include, for example, procedures for the supply of a source of chemically light-emitting energy, the mixture of the luminous substance with a labelling substance, and the removal of the labelling substance after luminescence in order to prevent background noise from being caused by residual substances.
The electrophoresis methods can be applied to, for example, the diagnosis of hereditary diseases, the investigation of DNAs in determining suspects of crimes and the investigation of the relationship between a parent and a child, in addition to the determination of the DNA sequence. In the diagnosis of hereditary diseases, it is currently possible to distinguish even one base from samples on the basis of the difference between the electrophoresis patterns by taking advantage of the difference in the structure of a high dimension under specific conditions (e.g. time or pH as causing a minute difference in denatured states of DNA) by the substitution of the base or bases inherent in each hereditary disease, such as single strand conformation polymorphism. On the other hand, the investigation of DNA in, for example, determining a suspect of crime and a parent-child relationship is made by comparing the difference in electrophoresis distances by taking advantage of a deviation in DNAs (polymorphism) between individuals.
In such experiments, the base length of a DNA is approximately 1,000 bases or less in many cases, and the gel material to be frequently employed for electrophoresis is polyacrylamide gel. In the case of the base length of a DNA of several thousands of bases, agarose gel is usually employed. Further, a gel electrophoresis apparatus of a flat plate type is employed for the comparison of the electrophoresis pattern of a sample with a reference DNA electrophoresis pattern. With such a gel electrophoresis apparatus, the sample and the reference DNA sample are subjected to gel electrophoresis side by side for a ready reference to the difference between the two electrophoresis patterns.
These methods, however, require care in, for example, sustaining homogeneity of a gel material with high stability and maintaining the uniformity of temperature on the electrophoresis plates during electrophoresis processes. In particular, very careful management of temperature using a thermostat is required in single strand conformation polymorphism. The management of temperature makes the cost of a device expensive and its size large because the electrophoresis apparatus of a flat plate type consumes a large amount of power and the amount of heat evolved is great. On the other hand, such problems inherent in the electrophoresis apparatus of the flat plate type can be solved by an electrophoresis apparatus of a capillary type because such a capillary-type electrophoresis apparatus can make its electrophoresis section smaller in size and it can be handled in a manner easier than that of the flat plate type.
However, the prior art electrophoresis apparatus so adapted as to read the electrophoresis pattern obtained by the fluorescence detecting method of conventional technology requires the use of a laser light source of a unique type corresponding to the wavelength at which the fluorescent substance is to be excited. The conventional electrophoresis apparatus suffers from various disadvantages. The cost of the laser light source, which accounts for most of the total cost of the apparatus, is so great that the cost of the apparatus itself becomes expensive as well. Further, laser light should be handled with great care because the laser light emitted from the laser light source has a high energy density even if it would scatter, so that there is the risk of causing disorders or abnormality of vision, such as dyschromatopsia or blindness, if the laser light would enter the naked eye. Hence, such a laser light source is to be incorporated in the electrophoresis apparatus with great attention paid to security from such laser light. This also leads to making the electrophoresis apparatus expensive.
The primary object of the present invention is to provide an apparatus for reading an electrophoresis pattern, which can be prepared at cheaper costs and handled in an easier fashion than conventional electrophoresis apparatuses.
The present invention has another object to provide a method for reading an electrophoresis pattern which allows for easier handling than conventional electrophoresis apparatuses.
The present invention has a further object to provide an apparatus for reading an electrophoresis pattern, which does not require an expensive device structure, including the use of a laser light source of a unique type.
The present invention has a still further object to provide a method for reading an electrophoresis pattern, which can read the electrophoresis pattern with higher sensitivity to detection of a sample than conventional electrophoresis apparatuses.
In order to achieve the objects as described hereinabove, the present invention consists of an apparatus for reading an electrophoresis pattern, which comprises: a sample supplying means for supplying a sample labelled with a fluorescent substance from an inlet side of an electrophoresis gel unit; an electrophoresis means for subjecting the sample to electrophoresis by applying an electrophoresis voltage to the electrophoresis gel unit; a carrying means for withdrawing the electrophoresed sample continually from an outlet side of the electrophoresis gel unit into a carrying fluid and carrying the carrying fluid a predetermined distance; a mixing means for mixing the carrying fluid with a luminous liquid or solution; and a light receiving means for receiving fluorescence emitted from the sample in the luminous liquid at a position to which the carrying fluid has been carried or transferred the predetermined distance.
In another aspect of the present invention, the apparatus for reading the gel electrophoresis pattern further comprises a bypass means for removing gases generated from an electrophoresis electrode disposed at the outlet side of the electrophoresis gel unit by bypassing a flow path through which the carrying fluid passes.
In a further aspect of the present invention, the gel electrophoresis pattern reading apparatus is arranged such that the carrying fluid is a liquid having a composition equivalent or similar to a buffer solution placed at the electrode side on the inlet side of the electrophoresis gel unit, wherein the luminous liquid or solution is capable of causing the fluorescent substance of the sample to emit fluorescence upon a chemical reaction with a peroxalic acid.
In a still further aspect of the present invention, the apparatus for reading the gel electrophoresis pattern comprises: an electrophoresis gel unit which is filled with gel material and in which electrophoresis is to be conducted; a first fine tube which is filled with the gel unit; a buffer solution container or tank for storing a buffer solution disposed so as for the buffer solution to come in contact with each of both ends of the gel unit disposed in the first fine tube; an electrode disposed so as to come into contact with the buffer solution in the buffer solution container or tank; a power source for applying electrophoresis voltage to the electrode; a second fine tube for carrying or transferring the electrophoresed sample separated while supplying the buffer solution; a third fine tube for supplying a luminous liquid; a mixer for mixing the solution withdrawn from the second fine tube with the liquid supplied from the third fine tube; a fourth fine tube for withdrawing the mixture from the mixer and allowing a chemical reaction to be carried out with the luminous liquid for a predetermined period of time so as to emit fluorescence to a sufficiently high intensity; a fluorescence detector disposed at a terminal end portion of the fourth fine tube for detecting the fluorescence emitted from the mixture; and a data processing unit for processing electrical signals converted from the fluorescence detected by the fluorescence detector.
In an additional aspect, the present invention consists of a method for reading an electrophoresis pattern, which comprises: supplying a sample labelled with a fluorescent substance from an inlet side of the electrophoresis gel unit; subjecting the sample to electrophoresis by applying an electrophoresis voltage to the electrophoresis gel unit; withdrawing the electrophoresed sample continually from an outlet side of the electrophoresis gel unit into a carrying fluid and carrying or transferring the carrying fluid a predetermined distance; mixing the carrying fluid with a luminous liquid; and receiving fluorescence emitted from the sample upon a chemical reaction in the luminous liquid at a position to which the carrying fluid has been carried the predetermined distance.
With the arrangement of the apparatus for reading the gel electrophoresis pattern in accordance with the present invention, the electrophoresis means is arranged to subject the sample to electrophoresis by applying the electrophoresis voltage to the electrophoresis gel unit when the electrophoresis gel unit is supplied with the sample labelled with a fluorescent substance by the sample supplying means. Further, the carrying means is arranged for withdrawing the electrophoresed sample into the carrying fluid continually from the outlet side of the electrophoresis gel unit and carrying the withdrawn fluid. The carrying fluid containing the electrophoresed sample, which is carried or transferred by the carrying means, is then mixed with the luminous liquid by the mixing means. In the carrying fluid containing the electrophoresed sample, an intermediate active substance or material produced as a result of a chemical reaction with the luminous liquid can excite the fluorescent substance labelled in the sample, thereby causing fluorescence to emit from the sample. The light receiving means is so disposed as to receive the fluorescence at the position to which the carrying fluid is carried or transferred over a predetermined period of time, until the fluorescent substance is excited so as to emit a sufficiently high intensity of fluorescence, in the path through which the carrying fluid is transferred and fluorescence is caused to be emitted from the fluorescent substance, that is, to which the carrying fluid is transferred a predetermined distance.
In the arrangement of the apparatus for reading the gel electrophoresis pattern in accordance with the present invention, no laser light is employed as the light for exciting the fluorescent substance of the electrophoresed sample and, for example, the intermediate active substance or material produced as a result of a chemical reaction with the luminous liquid capable of luminescence is employed in place of such laser light. The energy of the intermediate active substance or material is transmitted to the fluorescent substance and can exert the action of exciting the fluorescent substance in the sample to a sufficient extent. As a result, the fluorescent substance is excited, thereby evolving fluorescence to an intensity high enough to allow a detector of usual type to detect the electrophoresis fluorescence pattern with sufficiently high sensitivity.
The apparatus for reading the gel electrophoresis pattern according to the present invention may be provided with the bypassing means which in turn is so adapted as to remove the gases generated from the electrophoresis electrode on the outlet side of the electrophoresis gel unit by bypassing the path through which the carrying fluid flows. When the electrophoresis voltage is applied to the electrophoresis gel unit in order to subject the sample to electrophoresis, the gases are produced as a result of electrophoresis. If they are accumulated on the outlet side of the electrophoresis gel unit, the gases may act as a factor adversely affecting the electrophoresis so that they are required to be removed by the bypassing means. Further, the gases may disturb the flow of the carrying fluid in withdrawing the electrophoresed sample continually from the outlet side of the electrophoresis gel unit by means of the carrying means. Hence, the gases may act as a factor that disturbs the conveyance of the electrophoresed sample together with the carrying fluid so that the gases are to be removed by the bypassing means before the carrying fluid is carried.
The carrying fluid to be employed for this invention may be a liquid having a composition substantially identical to or similar to that of the buffer solution to be supplied from the inlet side of the electrophoresis gel unit. Hence, the carrying fluid can be utilized, too, as the buffer solution to be supplied to the inlet side of the electrophoresis gel unit. In this case, the electrophoresed sample separated from the gel unit is discharged from the outlet side of the electrophoresis gel unit into the carrying fluid. Then, the carrying fluid containing the electrophoresed sample is then carried or transferred intact.
For the apparatus for reading the gel electrophoresis pattern in accordance with the present invention, there may be employed, as the luminous liquid, a liquid capable of a chemical reaction with a peroxalic acid ester. The peroxalic acid ester can produce the intermediate active substance or material during the reaction with the fluorescent substance and can provide the fluorescent substance with its energy to thereby emit fluorescence. The amount or extent of the emission of fluorescence can readily be adjusted by adjusting the reaction of producing the intermediate active substance or material, and the amount of fluorescence for exciting the fluorescent substance can be adjusted by adjusting the reaction time.
In the method for reading the gel electrophoresis pattern in accordance with the present invention, the sample labelled with the fluorescent substance is supplied from the inlet side of the electrophoresis gel unit, the sample is subjected to electrophoresis by applying the electrophoresis voltage to the electrophoresis gel unit, the electrophoresed sample is withdrawn from the outlet side of the electrophoresis gel unit continually into the carrying fluid, the carrying fluid is then mixed with the luminous liquid, the carrying fluid is transferred the predetermined distance, and the fluorescence emitted from the sample in the carrying fluid is received.
Hence, the method according to the present invention can continually carry out the process for supplying the sample labelled with the fluorescent substance to the electrophoresis gel unit, the process for subjecting the sample to electrophoresis in the electrophoresis gel unit, the process for exciting the fluorescent substance in the sample electrophoresed in and separated from the electrophoresis gel unit, and the process for receiving fluorescence emitted from the fluorescent substance of the sample while transferring the carrying fluid containing the sample. The arrangement of the method can continually read electrophoresis patterns of the sample.
Other objects, features, and advantages of this invention become apparent in the course of the description of the preferred embodiments which follows, with reference to the accompanying drawings.